Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1

Neuronal activity is emerging as a driver of central and peripheral nervous system cancers. Here, we examined neuronal physiology in mouse models of the tumor predisposition syndrome Neurofibromatosis-1 (NF1), with different propensities to develop nervous system cancers. We show that central and peripheral nervous system neurons from mice with tumor-causing Nf1 gene mutations exhibit hyperexcitability and increased secretion of activity-dependent tumor-promoting paracrine factors. We discovered a neurofibroma mitogen (COL1A2) produced by peripheral neurons in an activity-regulated manner, which increases NF1-deficient Schwann cell proliferation, establishing that neurofibromas are regulated by neuronal activity. In contrast, mice with the Arg1809Cys Nf1 mutation, found in NF1 patients lacking neurofibromas or optic gliomas, do not exhibit neuronal hyperexcitability or develop these NF1-associated tumors. The hyperexcitability of tumor-prone Nf1-mutant neurons results from reduced NF1-regulated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel function, such that neuronal excitability, activity-regulated paracrine factor production, and tumor progression are attenuated by HCN channel activation. Collectively, these findings reveal that NF1 mutations act at the level of neurons to modify tumor predisposition by increasing neuronal excitability and activity-regulated paracrine factor production.

Neuronal activity is emerging as a driver of central and peripheral nervous system cancers. Here, we examined neuronal physiology in mouse models of the tumor predisposition syndrome Neurofibromatosis-1 (NF1), with different propensities to develop nervous system cancers. We show that central and peripheral nervous system neurons from mice with tumor-causing Nf1 gene mutations exhibit hyperexcitability and increased secretion of activity-dependent tumor-promoting paracrine factors. We discovered a neurofibroma mitogen (COL1A2) produced by peripheral neurons in an activity-regulated manner, which increases NF1-deficient Schwann cell proliferation, establishing that neurofibromas are regulated by neuronal activity. In contrast, mice with the Arg1809Cys Nf1 mutation, found in NF1 patients lacking neurofibromas or optic gliomas, do not exhibit neuronal hyperexcitability or develop these NF1-associated tumors. The hyperexcitability of tumor-prone Nf1-mutant neurons results from reduced NF1-regulated hyperpolarization-activated cyclic nucleotidegated (HCN) channel function, such that neuronal excitability, activity-regulated paracrine factor production, and tumor progression are attenuated by HCN channel activation. Collectively, these findings reveal that NF1 mutations act at the level of neurons to modify tumor predisposition by increasing neuronal excitability and activity-regulated paracrine factor production.
In addition to the critical contributions from immune system cells, we have recently shown that NF1 mutation in neurons synergizes with light-induced retinal ganglion cell activity to regulate neuroligin-3 (NLGN3) shedding and Nf1-optic pathway glioma (Nf1-OPG) initiation and growth 22 . This finding builds upon prior reports establishing that neurons and neuronal activity increase high-grade glioma growth through the secretion of paracrine factors, like NLGN3 and brain-derived neurotrophic factor (BDNF), in an activity-dependent manner 23,24 or by forming bona fide AMPA receptor-dependent neuron-to-glioma synapses 25,26 . Moreover, these effects of neuronal activity on high-grade glioma growth are amplified by glioma-induced hyperexcitability of neurons [26][27][28][29][30][31] .
To further elucidate the contribution of neuronal activity to central and peripheral nervous system tumor development, we focused on NF1, where affected individuals are prone to developing tumors intimately associated with nerves, including OPGs and pNFs [32][33][34] . Using these preclinical models, we previously demonstrated that different germline Nf1 mutations have dramatically different effects on plexiform neurofibroma and OPG formation in mice 17,35,36 , suggesting that the specific NF1 germline mutation may regulate tumorigenesis at the level of non-neoplastic cells.
In this study, we leveraged a common, naturally occurring NF1 missense mutation (c.5425C > T; p.Arg1809Cys) found in patients with NF1 who do not develop OPGs or neurofibromas. Exploiting this unique mutation, we employed a combination of human-induced pluripotent stem cell (hiPSC) and Nf1-mutant mouse lines to demonstrate that central (retinal ganglion cells; RGCs) and peripheral (sensory neurons and dorsal root ganglion cells; DRGs) nervous system neurons support tumor growth by secreting paracrine factors necessary for tumor progression in an Nf1 mutation-and neuronal activity-dependent manner. In contrast to mice with other NF1 patient germline NF1 gene mutations, mice with the Arg1809Cys mutation, like NF1 patients with this mutation, do not form pNFs or OPGs and their DRGs and RGCs, respectively, do not exhibit the RAS-independent neuronal hyperexcitability seen in tumor-forming Nf1-mutant central and peripheral nervous system neurons. Based on prior studies revealing that the NF1 protein, neurofibromin, binds to and regulates hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 37 and that HCN channels directly modulate neuronal excitability 38,39 , we now show that HCN channel dysregulation is responsible for Nf1-mutant central and peripheral nervous system neuronal hyperexcitability and consequently increased tumor-driving paracrine factor release, such that HCN channel targeting (using the anti-seizure medication lamotrigine) blocked Nf1-OPG progression in vivo. Moreover, we demonstrate that tumor-causing Nf1 mutations in neurons regulate neuronal production of paracrine factors through both visual experience (light)-evoked neuronal activity, as well as HCN channel dysregulation-mediated baseline neuronal hyperexcitability, highlighting the essential role of neuronal activity in NF1associated nervous system tumor progression.
OPG-associated Nf1-mutant CNS neurons are hyperexcitable. Prior studies from our laboratories have shown that OPG growth in Nf1-mutant mice (Nf1 f/neo ; hGFAP-Cre) is initiated by neuronal activity-dependent paracrine signaling 22 . In these mice, neuroligin-3 (Nlgn3) is shed in the Nf1-mutant (Nf1 +/neo ) optic nerve in an activity-dependent manner, such that genetic or pharmacological blockade of Nlgn3 shedding inhibits glioma initiation and progression 22 . Based on these findings, we first examined the neuronal activity of primary WT, Nf1 +/neo , and Nf1 +/1809 RGCs using multi-electrode arrays ( Fig. 2A) or calcium imaging (Fig. 2B) after 10 days in vitro. We found that the Nf1 +/neo , but not the Nf1 +/1809 , neurons had increased activity relative to WT RGCs, as measured by action potential (AP) firing rates (2.5-3.9-fold increase relative to WT control; Fig. 2A, B). No change in neuronal action potential amplitudes were noted in Nf1 +/neo or Nf1 +/1809 neurons relative to WT controls (Fig. 2C).
This suggests that Nf1 mutations associated with tumor formation cause RGC neurons to be hyperexcitable.
The correlation between neuronal midkine production and tumor risk is reinforced in human iPSC-derived central nervous system neurons ( Supplementary Fig. 2C). Midkine expression is increased both in excitatory (Fig. 2J) Fig. 2J, K) expression relative to WT controls, highlighting the selective upregulation of Nlgn3 and midkine in CNS, rather than in PNS, neurons.
As part of a neuron-immune-cancer cell axis in Nf1-OPG, Nf1mutant neurons secrete midkine to induce T-cell Ccl4 expression, which in turn, results in microglial elaboration of Ccl5, an obligate OPG growth factor 7,18,19 . To ascertain whether this molecular circuitry is intact in mice harboring the Nf1 +/1809 mutation, and to exclude defects in other stromal cells (T cells and microglia) that might be additionally responsible for the observed lack of optic gliomas in Nf1 f/1809 ; hGFAP-Cre mice, we examined the ability of Nf1 +/1809 T cells and microglia to secrete Ccl4 in response to midkine and Ccl5 in response to Ccl4, respectively ( Supplementary Fig. 2L, M). Both Nf1 +/1809 T cells and microglia responded to midkine and Ccl4, respectively, similar to their Nf1 +/neo counterparts 7 . Therefore, the lack of OPG formation likely reflects the failure of Nf1 +/1809 neurons to produce glioma-promoting trophic factors. Importantly, blockade of Nf1 +/neo neuronal activity with 1 µM tetrodotoxin (TTX) ( > 80-fold decrease; Fig. 2K, L) reduced midkine levels (1.9-fold decrease; Fig. 2M), similar to TTX effects on Nlgn3 22 , confirming that both Nlgn3 and midkine secretion are neuronal activitydependent and reversible by pharmacological treatment.
HCN channel activity regulates midkine production in OPGassociated Nf1 RGCs. To determine whether light-induced retinal ganglion cell neuronal activity regulates midkine secretion in the optic nerve, Nf1 +/neo mice were reared either in 12 h light/ dark cycles or completely in the dark for 4 weeks starting at 4 weeks of age. The retinae of dark-reared animals had decreased levels of Nlgn3 (48% decrease; Fig. 3A) relative to light/darkreared controls. In stark contrast, retinal Mdk RNA and protein expression were not affected by the decrease in visual experience (Fig. 3B, C), suggesting an alternative mechanism for neuronal activity-dependent midkine production.
Based on prior experiments demonstrating that HCN channels control neuronal hyperexcitability and that the Nf1 mutation regulates HCN channel function 37 , we examined the effect of Nf1 mutation on HCN channel function and neuronal excitability. Hcn1 and Hcn2 account for the majority of retinal Hcn channel expression; however, Nf1 mutation (Nf1 +/neo ) does not alter Hcn levels ( Supplementary Fig. 2N). To ascertain whether HCN channel function was responsible for the increased neuronal activity and Nlgn3/midkine production, we treated Nf1 +/neo RGC neurons with 200 µM lamotrigine (LTR), an HCN channel agonist, and assayed neuron activity for 3 min (Fig. 3D, E). Lamotrigine reduced the firing rates in Nf1 +/neo RGC neurons ( > 80% decrease; Fig. 3D, E). In striking contrast, while lamotrigine treatment of either heterozygous Nf1 +/neo or OPGbearing Nf1 f/neo ; hGFAP-Cre mice in vivo did not change Nlgn3 or Adam10 RNA expression (   Fig. 3H) relative to vehicle-treated controls. Conversely, treatment of WT and Nf1 +/1809 neurons with 30 µM of the HCN channel antagonist ZD7288 (ZD) resulted in a 14-15-fold increase in RGC neuron midkine production ( Fig. 3J) but did not alter Nlgn3 or Adam10 RNA expression (Fig. 3K). Identical results were obtained using hippocampal neurons (Supplementary Fig. 3A-H), supporting the idea that baseline neuronal hyperexcitability mediated by HCN function is a shared feature of Nf1-mutant CNS neurons. As a complementary genetic approach, we infected wild-type neurons using three separate short hairpins against Hcn1 and Hcn2. Both alone and in combination, infection of RGC and DRG neurons with the shHcn1/2 constructs resulted in rapid neuronal death within 6 hours ( Supplementary Fig. 4A, B), demonstrating that Hcn1 and Hcn2 presence is required for neuronal survival. Similarly, incubation of neurons with TTX, a drug that abolishes neuronal activity, also induces neuronal death within 6 hours( Supplementary Fig. 4C). Together, these data reveal the existence of an HCN channel-dependent mechanism for Nf1-mutant CNS tumor-associated neuronal midkine production.
Increased Nf1-mutant neuron activity is not RAS-dependent. As the NF1 protein (neurofibromin) functions a negative regulator of RAS activity (RAS-GTPase-activating protein), RAS-GTP levels were increased by 2.3-2.7-fold in Nf1 +/1809 RGC and hippocampal neurons relative to WT controls, similar to Nf1 +/neo neurons ( Fig. 3L and Supplementary Fig. 3I) and other mouse strains harboring NF1 patient-specific Nf1 germline mutations 47 . The finding of similarly increased RAS-GTP in Nf1 +/1809 CNS neurons suggests that RAS deregulation is not responsible for the failure of Nf1 f/1809 ; hGFAP-Cre mice to form tumors. However, it does not exclude RAS as a potential signaling effector downstream of HCN channel activity. In this respect, treatment of Nf1 +/neo neurons with the pan-RAS inhibitor, IN-1, reduced RAS-GTP levels ( Fig. 3M and Supplementary Fig. 3I), as well as midkine expression ( Fig. 3N and Supplementary Fig. 3J). In addition, systemic treatment of Nf1 +/neo ; hGFAP-Cre mice with the RAS inhibitor lovastatin decreased RGC midkine expression in vivo (Fig. 3O), indicating that RAS operates to control midkine expression. Conversely, whereas inhibition of Nf1 +/neo neuronal activity by TTX and lamotrigine reduced RAS hyperactivation ( Fig. 3P and Supplementary Fig. 3K), RAS (IN-1) inhibition had no effect on neuronal activity (Fig. 3Q, R and Supplementary  Fig. 3L). Taken together, these results position RAS-mediated neuron midkine production downstream of HCN channel activity, and demonstrate that increased baseline excitability of tumorassociated Nf1-mutant neurons is RAS-independent.
Increased HCN channel activity prevents OPG progression in vivo. To determine whether HCN channel function is critical for OPG formation, Nf1 f/neo ; hGFAP-Cre (Nf1-OPG) mice received intraperitoneal injections of lamotrigine from 6 to 8 weeks of age, at the time of early tumor evolution. Consistent with neuronal activity mediating Nf1-OPG progression, HCN activation by lamotrigine reduced OPG development at 3 months of age. Lamotrigine treatment did not decrease optic nerve volumes (1.5-fold increased volumes relative to WT controls; Fig. 3S), unlike dark-reared Nf1-OPG mice or those genetically lacking Ngln3 22 , where tumor initiation was completely prohibited. However, lamotrigine treatment resulted in reduced optic nerve proliferation (%Ki67 + cells; 5.7-fold decrease), as well as microglia (%Iba1 + cells; 1.7-fold decrease) and T-cell (CD3 + cells; 1.6-fold decrease) content, relative to vehicle-treated Nf1-OPG mice, comparable to WT mouse optic nerves (Fig. 3S, T). These results indicate that HCN channel-regulated midkine production is necessary for tumor progression, rather than initiation, but establish HCN channel activity as a targetable regulator of neuronal activity-dependent tumor progression.
Based on our findings in the CNS, we hypothesized that PNS tumor (plexiform neurofibroma) growth is also dependent upon neuron activity-dependent paracrine factor secretion. Since neuronal trophic factors that mediate plexiform neurofibroma preneoplastic cell (NF1 −/− Schwann cells; shNF1 SCs, Supplementary Fig. 1A) growth have not yet been identified, we leveraged hiPSC-derived sensory neurons that harbor heterozygous NF1 mutations found in patients with (c.1149 C > A, p.Cys381X; c.2041 C > T, pArg681X; c.6619 C > T, p.Gln2207X; Group 1) or without (c.5425 C > T; p.Arg1809Cys; Group 2) neurofibromas ( Fig. 5E and Supplementary Fig. 5B, C). As Schwann cells are the proliferative neoplastic cells in neurofibromas, their in vitro proliferation was used as a proof-ofprinciple measure of their potential to proliferate within a neurofibroma in vivo. We found that conditioned media (CM) from group 1, but from not group 2, NF1-mutant neurons increased preneoplastic shNF1 Schwann cell proliferation (3.4-3.6-fold increase in Ki67 + Schwann cells; Fig. 5E and Supplementary Fig. 5D).
Leveraging these observations, we performed unbiased protein secretome analyses on CM from control, and representative sensory neurons from group 1 (NF1 R681X ) and group 2 (NF1 R1809C ; Fig. 5F and Supplementary Fig. 6B, C). The secreted proteins from both NF1-mutant neurons were compared to those of the controls and each differentially regulated protein was assigned an arbitrary identification number. From the 176 differentially regulated proteins, the expression of six proteins was uniquely increased more than 1.5-fold in the tumorassociated NF1 R681X CM but not in the non-tumor-associated NF1 R1809C CM relative to control CM (Fig. 5F). As a secondary validation, CM from independently generated sensory neurons was used to confirm the presence and concentration of the six identified proteins. Of these, only COL1A2 was elevated in the CM from the tumor-associated group 1, but not in the nontumor-associated group 2, hiPSC-sensory neurons, as well as in mouse Nf1 +/neo but not Nf1 +/1809 DRG neurons (2.4-3.2-fold increase; Fig. 5G, H and Supplementary Fig. 6D-H). Importantly, both Nf1 +/neo mouse DRG (Fig. 5I) and NF1 R681X hiPSC-sensory neuron CM (Supplementary Fig. 6I) increased Nf1 −/− DRG-NSCs (murine Schwann cell progenitors) proliferation (2-8-3.1fold increase in %Ki67 + cells) relative to control and Nf1 +/1809 or NF1 R1809C neuron CM. Notably, COL1A2 was uniquely expressed by NF1-mutant PNS, but not CNS, neurons (Supplementary Fig. 6J).
Finally, to determine whether HCN channel function can govern pNF progression in vivo, mice harboring NF1-pNFs  received intraperitoneal injections of lamotrigine for 6 weeks. HCN activation reduced pNF size, partly restored neuronal histology, and reduced both proliferation (Ki67 + cells), as well as Col1A2 immunoreactivity, within the tumors (Fig. 7J). Together, these data firmly establish that HCN channel-mediated sensory neuron Col1a2 production regulates pNF progression in vivo.

Discussion
Exploiting a unique, naturally occurring germline mutation in patients with the NF1 tumor predisposition syndrome who fail to develop neurofibromas or optic gliomas (Arg1809Cys), we employed hiPSCs and genetically engineered mice to identify two distinct mechanisms underlying neuronal activity regulation of nervous system tumor progression (Fig. 8).
In this study, and similar to what is observed in patients with NF1 40,44,45 , we first show that Nf1 +/1809 mice do not form pNFs or OPGs. Consistent with the lack of tumor formation, Arg1809Cys-mutant neurons do not induce Adam10-mediated cleavage and shedding of Nlgn3, a growth factor required for murine Nf1-OPG initiation and growth 22 . In addition, we previously described a neuron-immune-cancer cell axis 7 , where neurons indirectly regulate Nf1-OPG progression through their effects on T-cell Ccl4-mediated induction of microglial growth factor (Ccl5) production. Since Nf1 +/1809 T cells produce Ccl4 in response to midkine and Nf1 +/1809 microglia produce Ccl5 in response to Ccl4, the Arg1809Cys mutation appears to operate at the level of the neuron, such that human and mouse neurons with this mutation fail to increase midkine expression or activate optic glioma-infiltrating T cells to drive Nf1-OPG progression.
Second, we identified COL1A2 as a sensory neuron-derived paracrine factor important for NF1-deficient Schwann cell proliferation. Of note, Schwann cells are the neoplastic cells of two distinct types of tumors, neurofibromas, and schwannomas, which differ both in pathology and immunohistochemical profiles. Specifically, neurofibromas, which occur both sporadically and in the setting of NF1, are heterogeneous tumors with small and wavy nuclei, excess "shredded" type collagen, and are immunopositive for neurofilament expression. In contrast, schwannomas arising either sporadically or in patients with neurofibromatosis type 2 (NF2) and Schwannomatosis are encapsulated tumors with more homogeneous Schwann cell proliferation, larger nuclear sizes, and the presence of hyalinized vessels 54 . The importance of collagen to neurofibroma-associated Schwann cell growth is underscored by the observation that collagen accounts for the majority of the extracellular matrix in human neurofibromas and as much as 50% of neurofibroma dry weight 55 . While type 1 collagens increase Schwann cell and Schwann cell progenitor adhesion, survival, and proliferation 56-58 , we show that NF1 mutation in human and mouse peripheral sensory neurons induces activity-dependent production of COL1A2, which, similar to NLGN3 in the brain [22][23][24] , induces a feed-forward loop of COL1A2 transcription in Schwann cells and Schwann cell progenitors, resulting in elevated tumoral collagen levels. While the abundance of collagen and its production by other cell types (fibroblasts) in neurofibromas 11 prompted human clinical trials with broad-spectrum anti-fibrotic agents, like Pirfenidone, no efficacy was observed 59 , possibly due to the low abundance of collagensynthesizing fibroblasts in pNFs 60 . Ongoing studies are focused on determining whether targeting of sensory neuron-specific COL1A2 production will reduce neurofibroma growth.
Third, examination of NF1 +/1809 neurons revealed unique non-RAS functions for the NF1 protein, neurofibromin. In this regard, NF1 +/1809 neurons exhibit elevated RAS activity, similar to neurons with NF1 mutations from patients who develop neurofibromas or optic gliomas. However, Nf1 +/1809 neurons do not exhibit increased action potential firing rates necessary to drive Nlgn3 and midkine (retinal ganglion cells) or COL1A2 (sensory neurons) secretion. These findings uncouple RAS regulation from the control of baseline neuronal excitability, and suggest that other non-RAS-dependent mechanisms account for these neurofibromin-regulated effects in neurons. While there are a few examples of non-RASdependent functions for neurofibromin 37,47,61-63 additional studies will be necessary to determine whether the NF1 Arg1809Cys mutation, located within the PH-like domain of neurofibromin 64 , affects the conformation of the protein relative to neurofibromin dimerization 65 , secondary structure 40,64 , or associations with other neurofibromin-binding partners in neurons [66][67][68] .
Fourth, we demonstrate that NF1 mutation regulates neuronal hyperexcitability intrinsically through HCN channel function, and this hyperexcitability is evident in visual experience-evoked activation. The finding of hyperexcitability parallels prior studies of Nf1 +/neo sensory neurons, which have greater numbers of action potentials, lower firing thresholds, lower rheobase currents, and shorter firing latencies 69 . Herein, we demonstrate that baseline NF1 regulation of neuronal hyperexcitability involves dysregulated HCN channel function (midkine, COL1A2 production) 37 . HCN channels are voltage-operated cation channels expressed in RGC 70,71 . and DRG neurons [71][72][73] . Inhibition of HCN channel signaling with antagonists, such as ZD7288, increases neuron firing rates in vivo 74 , paralleling the effects of HCN channel agonist (LTR) and antagonist (ZD7288) treatments on CNS and PNS neuron hyperexcitability and activitydependent regulation of midkine and Col1a2 expression. Additionally, RGC hyperexcitability in the context of visual experience and consequent Adam10/Nlgn3 production are required for Nf1-OPG initiation, such that Nf1-optic glioma-prone mice do not develop tumors if reared in the dark during critical periods of tumorigenesis, or if Nlgn3 is genetically or pharmacologically blocked 22 . As light-induced activity did not affect RGC midkine expression and Adam10/Nlgn3 production was not dependent on HCN channel function, we postulate that Nf1-OPG initiation relies on light-mediated RGC activation and Nlgn3 shedding, whereas OPG progression requires both Nlgn3 shedding and HCN channel-regulated baseline neuronal activity and midkine production.
Taken together, the findings reported herein advance our growing appreciation of neurons as active participants in tumor biology. While we conclusively establish that neuronal hyperexcitability drives mouse Nf1 OPG and pNF progression, future work using genetically engineered mouse strains and ectopic gene delivery methods will be necessary to demonstrate that midkine and Col1a2 expression are solely sufficient to maintain murine OPG and pNF growth in vivo, respectively. Additional efforts will include the identification of key modulators of central and Fig. 5 pNF-associated NF1-mutant PNS neurons exhibit increased activity and COL1A2-dependent preneoplastic NF1 −/− Schwann cell growth. A, B Nf1 +/neo , but not Nf1 +/1809 , DRG neuron AP firing rates are elevated relative to WT DRG neurons, as measured by (A) multi-electrode array (WT, n = 24, Nf1 +/neo , n = 10; P = 0.0005, Nf1 +/1809 n = 10, ns), or (B) calcium imaging recordings (WT n = 8, Nf1 +/neo n = 5, P < 0.0001, Nf1 +/1809 n = 14, ns). C, D TTX (1 µM) and lamotrigine (LTR; 200 µM) reduce Nf1 +/neo DRG neuron AP firing rate as measured by multi-electrode array (vehicle n = 4, TTX n = 7, P < 0.0001; LTR n = 6, P < 0.0001) and calcium imaging (vehicle n = 23, TTX n = 9, P < 0.0001, LTR n = 14, P < 0.0001). The right panels show representative (A, C) spike plots of entire multi-electrode array well recordings over 30 s, and (B, D) traces of neuronal activity over 3 min. E Schematic illustrating treatment of human shNF1 Schwann cells with hiPSC-sensory neuron conditioned media (CM). NF1-deficient Schwann cell proliferation is increased after treatment with NF1 C383X , NF1 R681X , and NF1 E2207X mutant neuron CM (P < 0.0001), but not NF1 R1809C neuron CM relative to controls (CTL). n = 6 for all groups. F Analytical comparison of 2D gel electrophoresis (top-to-bottom: decreasing molecular weight; left-to-right: decreasing acidity) of NF1 R681X (left) and NF1 R1809C (right) CM relative to CTL hiPSC-sensory neuron CM. Red dots indicate proteins with increased expression, green dots indicate proteins with decreased expression, and yellow dots indicate unaltered proteins in NF1-mutant sensory neuron CM relative to CTL neuron CM. The six proteins uniquely increased more than 1.5-fold in NF1 R681X hiPSC-sensory neuron CM relative to CTL, but not in NF1 R1809C CM, relative to CTL are circled in blue and are listed in the lower panel. Representative CM from CTL, NF1 R1809C , and NF1 R681X sensory neurons was analyzed by 2D gel electrophoresis (n = 1). G, H COL1A2 levels are increased in (G) NF1 C383X , NF1 R681X , and NF1 E2207X mutant neuron CM (P < 0.0001), but not in NF1 R1809C neuron CM (n = 4 for all groups), as well as in (H) Nf1 +/neo mouse DRG neuron CM (P < 0.0001), but not in Nf1 +/1809 mouse DRG neuron CM (n = 6 for all groups). I Nf1-deficient DRG-NSC proliferation is increased after treatment with Nf1 +/neo DRG neuron CM (P < 0.0001), but not Nf1 +/1809 DRG neuron CM, relative to WT controls. n = 6 for all groups. Data are presented as the mean ± SEM. A-E, G-I One-way ANOVA with (A-D, G-I) Dunnett's, or (E) Tukey's multiple comparisons test. P values are indicated within each panel. ns, not significant. Source data are provided as a Source Data file.
peripheral nervous system neuron-dependent tumorigenesis. This presents unique opportunities to repurpose FDA-approved compounds that target neuron-produced mitogens (e.g., collagenase 75 ) or HCN channels (e.g., Lamotrigine; Ivabradine 76,77 ) for the treatment of NF1-associated nervous system tumors, expanding the toolbox for targeting neuron-low-grade tumor interactions in cancer.

Methods
All experiments were performed in compliance with active Animal Studies Committee protocols at Washington University and UT Southwestern.

Mice. All experiments were performed under active Animal Studies Committee protocols at Washington University School of Medicine (Washington University in St
Louis Institutional Animal Care and Use Committee) and UT Southwestern (UT Southwestern Institutional Animal Care and Use Committee). According to these ethics committees, any animals with compromised motion/eating habits or an unhealthy appearance are euthanized. No animals were euthanized due to their tumor burden or as a result of the treatments performed in this study. Mice were maintained on a 12 light/ dark cycle in a barrier facility, at 21°C and 55% humidity, and had ad libitum access to food and water. Heterozygous Nf1 c.5425 C > T; Arg1809Cysmutant mice were generated by CRISPR/ Cas9 engineering directly into C57Bl/6J embryos, resulting in mice with one wild-type Nf1 allele and one missense R1809C mutation. The mutation was confirmed by direct sequencing (IDT Technologies). R1809C Nf1-mutant mice, as well as heterozygous R681X 35 Human-induced pluripotent stem cells and neuronal differentiation. NF1 patient heterozygous germline NF1 gene (Transcript ID NM_000267) mutations were CRISPR/Cas9-engineered into a single commercially available male control human iPSC line (BJFF.6) by the Washington University Genome Engineering and iPSC Core Facility (GEiC). hiPSCs were authenticated based on morphology, as well as by immunocytochemical expression of pluripotency markers. Human iPSCs were differentiated into neural progenitor cells after 7 days of embryoid body formation (StemDiff Neural induction media; STEMCELL Technologies), followed by embryoid body dissociation and plating in PLO/Laminin-coated flasks in 50% DMEM/F12, 50% Neurobasal medium supplemented with N2, B27, 2 mM Glu-taMAX (all Gibco), 10 ng/mL hLIF, 3 μM CHIR99021 and 2 μM SB431541 (all STEMCELL Technologies). NPCs were subsequently differentiated either into excitatory CNS neurons following incubation in neurobasal medium supplemented with B27, 2 mM glutamine, and 50 U/mL penicillin/streptomycin for a minimum of 2 weeks, or into GABAergic CNS neurons following incubation in neurobasal medium supplemented with 1 μM cAMP, 10 ng/mL BDNF, 10 ng /mL GDNF, and 10 ng/mL IGF1 47 . For sensory neuron differentiation, iPSCs were incubated for 8 days in DMEM/F12 supplemented with LDN-193189, CHIR99021, A83-01, RO4929097, SU5402, retinoic acid, and 10% knockout serum replacement followed by 4 weeks of neurobasal medium supplemented with NT3, nerve growth factor, brain-derived neurotrophic factor, and glial-derived neurotrophic factor. No commonly misidentified cell lines were used in this study.
Spinal cord dissection and optic nerve processing. Mice were transcardially perfused at 3 months of age with Ringer's solution and 4% paraformaldehyde. Whole spinal cords were isolated following the removal of gross and muscle tissue and the breaking of vertebral column bones under a microdissection microscope. The entire spinal cord and peripheral nerves were rinsed and fixed in 10% formalin-buffered solution. DRG diameters were measured as previously reported 53,80 and tumor volumes were calculated as volume = length × width 2 × 0.52, which approximates the volume of a spheroid 53,80 . Optic nerves were isolated, imaged using a Leica DFC 3000 G camera, and their volumes were calculated as previously described 81 . Using ImageJ, four diameter measurements were taken to estimate the thickness of each optic nerve beginning at the chiasm (D 0 ), at 150 (D 150 ), 300 (D 300 ), and 450 µm (D 450 ) anterior to the chiasm. The following equation was used to calculate the estimated optic nerve volume in each of the three sections, the sum of which was ultimately used to calculate the total optic nerve volume: V 1 = 1/12 πh (D 0 2 + D 0 D 150 + D 150 2 ).
T cell and microglia isolation. Four to six-week-old WT and Nf1 +/1809 mouse spleens were homogenized into single-cell suspensions by digestion in PBS containing 0.1% BSA and 0.6% sodium citrate. The homogenates were subsequently washed and incubated with 120 Kunitz units of DNase I for 15 min following red blood cell lysis (eBioscience). Cells were then filtered through a 30 µM cell strainer to obtain a single-cell suspension. T cells were maintained at 2.5 × 10 6 cells ml −1 in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. T cells were treated with 100 ng/µL midkine (R&D Systems) for 48 h. Microglia isolation was performed on 4-6-week-old WT and Nf1 +/1809 mouse brains using the multi-tissue dissociation kit (Miltenyi Biochemicals) following published protocols 7 . The resulting cells, microglia attached to a monolayer of astrocytes, were maintained in minimal essential medium supplemented with 1 mM L-glutamine, 1 mM sodium pyruvate, 0.6% D-( + )-glucose, 1 ng/ml GM-CSF, 100 μg/ml P/S, and 10% FBS. Sensory neuron conditioned media protein analysis and validation of candidate proteins. Control, NF1 +/R1809C or NF1 +/R681X sensory neurons were washed with PBS and were incubated with artificial cerebral spinal fluid (aCSF) for 24 h prior to collecting conditioned media (CM). The media was treated with protease inhibitors (Cell Signaling Technologies), was snap frozen, and sent to Applied Biomics for 2D gel electrophoresis analysis. The conditioned media was run on a 2D electrophoresis gel and the proteins were separated by size and pH, as per the vendor's specifications. The resulting digital images of the 2D gels of CTL, NF1 +/R681X and NF1 +/R1809C conditioned media ( Supplementary Fig. 6B) were digitally superimposed pairwise by Applied Biomics (CTL vs NF1 +/R681X and CTL vs NF1 +/R1809C ) in order to detect differentially expressed proteins between each of the NF1-mutant neurons and the controls. In total, 176 dots (proteins) were upregulated or downregulated more than 1.5-fold relative to the CTL conditioned media, each dot was assigned a random identification number, and the intensity of the relative expression of each protein was translated into numerical values by the vendor. From the 176 differentially regulated proteins, only six (circled in blue; Fig. 5E) were upregulated more than 1.5-fold in NF1 +/R681X but not NF1 +/R1809C relative to CTL sensory neuron CM. As such, the identity of these six proteins alone was determined by mass spectrometry by Applied Biomics, following vendor specifications (Source Fig. 6 COL1A2 is necessary and sufficient for NF1-deficient Schwann cell growth in vitro. A Immunofluorescent staining and corresponding quantitation of Ki67 + human shNF1 Schwann cells (left) and Nf1 −/− mouse DRG-NSCs (right) following incubation with hiPSC-sensory neuron conditioned media (CM), with (h P = 0.0007; m P < 0.0001) and without (P < 0.0001) collagenase (n = 6 for all groups), COL1A2 alone with (h P = 0.0036; m P < 0.0001) and without (P < 0.0001) collagenase (n = 6 for all groups), as well as with and without control or short hairpins against COL1A2 (n = 3 for all groups, P < 0.0001) or Col1a2 (vehicle n = 4, control short hairpin n = 7, shCol1a2-1 n = 4, sh Col1a2-2 n = 4, sh Col1a2-3 n = 3, P < 0.0001). B-C (B) Human and (C) mouse cutaneous (cNF) and plexiform neurofibromas (pNF) express COL1A2. Normal brain, lymph node and normal sural (human) or normal sciatic (mouse) nerves were negative for COL1A2 expression. Neurofilament was used as positive control for normal mouse nerve tissue. These data derive from a single-tissue microarray. D COL1A2 RNA expression is increased in human shNF1 Schwann cells (left; P = 0.0014) and mouse Nf1 −/− DRG-NSCs (right; P = 0.0012) following COL1A2 treatment. n = 3 for all groups. E COL1A2 RNA expression is increased in human Schwann cells isolated from human cNF (P = 0.0039) and pNF tumors (P = 0.0022) relative to controls. Normal n = 10, cNF n = 11, pNF n = 11. Data are presented as the mean ± SEM. A, E Oneway ANOVA with (A) Tukey's or (E) Dunnett's multiple comparisons test, or (D) paired two-tailed Student t test. Scale bars, 50 µm. Source data are provided as a Source Data file.
data). No large-scale mass spectrometry or raw proteomics data was generated for these analyses. The concentration of each of these six identified proteins was assayed in independently generated CTL and NF1-mutant Schwann cell growth-promoting (NF1 C383X , NF1 R681X , NF1 E2207X ) and NF1-mutant non-Schwann cell growthpromoting (NF1 R1809C ) sensory neurons ( Fig. 5F and Supplementary Fig. 6D  plated in a minimum of six individual wells, with a minimum of three wells serving as the vehicle-treated controls and a minimum of three wells as the treated cohort. All efforts were taken to ensure even spreading of the neurons throughout each well. Not all 16 electrodes present within each well were within the optimal proximity to neurons and as such not all electrodes detected action potentials (APs). To account for this variation, all metrics were normalized to the number of the active electrodes only. In addition, as the number of active electrodes/well varied between technical replicates of each animal, the AP firing rate of all the replicate wells of each animal was averaged. As such, each data point graphed represents the average of all technical replicates for each given animal. All neurons were recorded for 3 min at a 4.5 standard deviation threshold level and 5000 Hz as a digital filter using AXION Biosystems integrated studio (AxIS) version 2.5.1 software. AP firing rates were calculated from the total number of APs/3 min and are represented as APs/min, only accounting for active electrodes. Representative traces of action potentials were extracted using the AXION Biosystems neural metric tool and Offline sorter x64 version 4 software.
Calcium imaging of neurons. Primary RGC (150,000 neurons/well) or DRG (75,000 neurons/well) neurons were plated onto poly-D-lysine and laminin-coated 96-well plates for 10 days. At 10 days, the cells were treated with Fluo-8/AM (1345980-40-6, AAT Bioquest), PowerLoad (P10020, ThermoFisher) and Probenecid (P36400; ThermoFisher) for 30 min at 37°C and for another 30 min at room temperature. The neurons were subsequently washed with HBSS and incubated for a minimum of 10 min in fresh culture medium supplemented with 5% neuro-background suppressor (F10489; ThermoFisher). The neurons were imaged on a Nikon spinning disk upright epi-fluorescence confocal microscope equipped with a ×10 dry objective, and a 488 nm wavelength laser was used for wide-field imaging. The neurons were stimulated by a Ti LAPP DMD (Deformable Mirror Device) LED source for ultrafast photo-stimulation, with 0.1 mW applied during each recording for Fluo-8 excitation. Fluo-8 images were collected at 15 Hz (2048 × 2048 pixels, 1 × 1 mm) and the duration of each region of interest (ROI) was limited to 10 min. The fluorescence intensity and optical response to depolarizing membrane potential transients (ΔF/F) were calculated in Matlab programming environment to generate single-neuron activity traces. The ΔF/F threshold was set at 4 standard deviation beyond baseline fluorescence. Following data acquisition, the duration and shape of each AP spike were compared by merging all the spikes in the same time window. Neurons from each animal were seeded in six wells and a minimum of three neurons were recorded per well. Data recorded from a minimum of 18 neurons per animal were averaged. Each data point represents a single animal.
Immunohistochemistry and Immunocytochemistry. All spinal cord and optic nerve fixed tissues as well as human brain tissue, lymph nodes, normal sural nerve, cutaneous neurofibromas or plexiform neurofibromas, and mouse sciatic nerve, cutaneous or plexiform neurofibromas were paraffin-embedded, serially sectioned (5 μm) and immunostained with GFAP, Iba1, Ki67, CD3, Midkine, GAP43, CD34, Factor XIIIa, SOX10, neurofilament-200, and Col1a2 (Supplementary Table 1  , which drives OPG initiation and cell growth. Second, tumor-associated NF1-mutant RGCs have increased intrinsic baseline neuronal hyperexcitability, which is controlled by HCN channel function. Increased baseline HCN channel-regulated RGC excitability triggers increased midkine production to induce a T-cell (Ccl4) and microglial (Ccl5) signaling cascade that governs OPG progression and growth. PNS, peripheral nervous system, CNS, central nervous system, pNF, plexiform neurofibroma, OPG, optic pathway glioma. Small elements of this schematic were designed on BioRender.com. Source data are provided as a Source Data file.
Western blotting. Western blotting was performed on snap-frozen cells and tissues. Samples were lysed in RIPA buffer (Fisher) supplemented with a protease inhibitor cocktail (Cell Signaling) and were blotted using appropriate primary (s-Nlgn3, neurofilament-200, peripherin, BRN3A, ISL1, CALCA, α-tubulin, β-actin; Supplementary Table 1) and NIR-conjugated secondary antibodies (Licor). Images were captured and analyzed using the Li-Cor Image Studio Lite Version 5.2 software and are representative of more than three independently generated biological replicates.
Quantitative real-time PCR. Total RNA was extracted following the manufacturer's instructions (QIAGEN) and reverse-transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) qPCR was performed using TaqMan gene expression assays (Mdk, Col1a2, COL1A2, Nlgn3, Adam10, NLGN3, ADAM10, Hcn1-4; Supplementary Table 2) and TaqMan Fast Advanced Master Mix (Applied Biosystems) according to the manufacturer's instructions. All reactions were performed using the Bio-Rad CFX96 Real-Time PCR system equipped with Bio-Rad CFX Manager 3.1 software. Gene expression levels of technical replicates were estimated by ΔΔCt method using GAPDH or Gapdh (Supplementary Table 2) as reference genes.
In vivo mouse lovastatin and lamotrigine treatments. In total, 17 Nf1 flox/neo ; GFAP-Cre (Nf1-OPG) mice were intraperitoneally administered vehicle (saline in 1% methylcellulose; n = 9) or 25 mg/ Kg body weight lamotrigine (Selleckchem; n = 8) from four to six weeks of age, three times a week. The mice were then aged to 12 weeks for optic nerve and RGC analysis. For lovastatin treatments, 20 Nf1-OPG mice were administered with 10 mg/kg/day lovastatin (Santa Cruz Biotechnologies; n = 10) or vehicle (saline in 1% methylcellulose; n = 10) by oral gavage for 4 weeks, 5 days a week, beginning at 12 weeks of age, for 8 weeks. The mice were analyzed at 20 weeks of age. For pNF analyses, Nf1 −/− DRG-NSCs were implanted in sciatic nerves of 8week-old athymic nude mice as previously described 49 . Briefly, the mice underwent surgery to create a pocket by displacing the quadriceps muscle and exposing their sciatic nerve. In all, 1 × 10 6 Nf1 −/− DRG-NSCs were implanted in the pocket around the sciatic nerve, such that the cells could be in direct contact with the nerve before the muscle and skin were sutured. Following recovery from the surgery, the mice were intraperitoneally administered vehicle (saline in 1% methylcellulose; n = 5) or 25 mg/kg body weight lamotrigine (Selleckchem; n = 5) three times a week for 6 weeks prior to histological analysis.
Published RNA database analysis. The analysis for this paper was generated using Partek Flow software, version 10.0 using publicly available datasets (GEO: GSE14038; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14038, Supplementary Table 3). RNA-seq reads were aligned to the Ensembl release 100 toplevel assembly with STAR version 2.7.8a. Gene counts and isoform expression were derived from Ensembl output. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. Normalization size factors were calculated for all gene counts by CPM to adjust for differences in sequencing depth. Genes not expressed on average with greater than two count-per-million were excluded from further analysis. Genespecific analysis was then performed using the lognormal with shrinkage model (limma-trend method) to analyze for expression differences between conditions. Short hairpin constructs, lentiviral production, and neuronal infection. Human shCOL1A2 and mouse shCol1a2 lentiviral particles (TRCN0000090043; TRCN0000090045; TRCN0000335210) were generated as previously described 61 . NF1 +/R681X or Nf1 +/neo sensory neurons were infected with three independent shCOL1A2 lentiviral particles or shRNA scrambled control particles (sc-108080; Santa Cruz Biotechnology) for 24 h. Neuronal media was refreshed and conditioned media was collected for subsequent assays 48-72 h post infection.
Quantification and statistical analysis. All statistical tests were performed using GraphPad Prism software (versions v5, v_8.2.1, and v_9.3.1). Paired or unpaired two-tailed Student's t tests or one-way analysis of variance (ANOVA) with Tukey's, Dunnett's, or Bonferroni post-test correction using GraphPad Prism 5 software. Statistical significance was set at P < 0.05, and individual p values are indicated within each graphical figure. A minimum of three independently generated biological replicates was employed for each of the analyses. Numbers (n) are noted for each individual analysis.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Publicly available RNA sequencing datasets (GEO: GSE14038) were analyzed in this study. Source data are provided with this paper.